The detection of nucleic acids is central to medicine, forensic science, industrial processing, crop and animal breeding, and many other fields. The ability to detect disease conditions (e.g., cancer), infectious organisms (e.g., HIV), genetic lineage, genetic markers, and the like, is ubiquitous technology for disease diagnosis and prognosis, marker assisted selection, identification of crime scene features, the ability to propagate industrial organisms and many other techniques. Determination of the integrity of a nucleic acid of interest can be relevant to the pathology of an infection or cancer.
One of the most powerful and basic technologies to detect small quantities of nucleic acids is to replicate some or all of a nucleic acid sequence many times, and then analyze the amplification products. Polymerase chain reaction (PCR) is a well-known technique for amplifying deoxyribonucleic acid (DNA). With PCR, one can produce millions of copies of DNA starting from a single template DNA molecule. PCR includes phases of “denaturation,” “annealing,” and “extension.” These phases are part of a cycle which is repeated a number of times so that at the end of the process there are enough copies to be detected and analyzed. For general details concerning PCR, see Sambrook and Russell, Molecular Cloning—A Laboratory Manual (3rd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (2000); Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (supplemented through 2005) and PCR Protocols A Guide to Methods and Applications, M. A. Innis et al., eds., Academic Press Inc. San Diego, Calif. (1990).
The PCR process phases of denaturing, annealing, and extension occur at different temperatures and cause target DNA molecule samples to replicate themselves. Temperature cycling (thermocyling) requirements vary with particular nucleic acid samples and assays. In the denaturing phase, a double stranded DNA (dsDNA) is thermally separated into single stranded DNA (ssDNA). During the annealing phase, primers are attached to the single stranded DNA molecules. Single stranded DNA molecules grow to double stranded DNA again in the extension phase through specific bindings between nucleotides in the PCR solution and the single stranded DNA. Typical temperatures are 95° C. for denaturing, 55° C. for annealing, and 72° C. for extension. The temperature is held at each phase for a certain amount of time which may be a fraction of a second up to a few tens of seconds. The DNA is doubled at each cycle, and it generally takes 20 to 40 cycles to produce enough DNA for certain applications. To have good yield of target product, one has to accurately control the sample temperatures at the different phases to a specified degree.
More recently, a number of high throughput approaches to performing PCR and other amplification reactions have been developed, e.g., involving amplification reactions in microfluidic devices, as well as methods for detecting and analyzing amplified nucleic acids in or on the devices. Thermal cycling of the sample for amplification is usually accomplished in one of two methods. In the first method, the sample solution is loaded into the device and the temperature is cycled in time, much like a conventional PCR instrument. In the second method, the sample solution is pumped continuously through spatially varying temperature zones. See, for example, Lagally et al. (Analytical Chemistry 73:565-570 (2001)), Kopp et al. (Science 280:1046-1048 (1998)), Park et al. (Analytical Chemistry 75:6029-6033 (2003)), Hahn et al. (WO 2005/075683), Enzelberger et al. (U.S. Pat. No. 6,960,437) and Knapp et al. (U.S. Patent Application Publication No. 2005/0042639).
Many detection methods require a determined large number of copies (millions, for example) of the original DNA molecule, in order for the DNA to be characterized. Because the total number of cycles is fixed with respect to the number of desired copies, the only way to reduce the process time is to reduce the length of a cycle. Thus, the total process time may be significantly reduced by rapidly heating and cooling samples to process phase temperatures while accurately maintaining those temperatures for the process phase duration.
The technique of melt analysis is becoming a standard tool for analyzing nucleic acid molecules following amplification. Melt analysis is also referred to in the art as high resolution melting (HRM), thermal melting, and melt curve analysis, and relies on the principles of the denaturing phase of amplification. That is, as a double stranded DNA (dsDNA) is subjected to increased temperatures, at a particularly temperature the dsDNA will be separated into single stranded DNA (ssDNA), thereby releasing any bound detection agents such as fluorescence markers, which can be optically detected and analyzed. These techniques are widely used, however, most systems rely on a heater block into which samples are inserted, spinning the sample tube/capillary through heated air, or establishing a temperature gradient that subjects the sample to different temperatures based on its position along the gradient. The temperature measurements are therefore based on measurement of the heater block, the air, or the opposite ends of the temperature gradient.
For instance, U.S. Pat. No. 7,785,776 from Idaho Technology, Inc., and the University of Utah Research Foundation describes at column 19 how “the high-resolution instrument also ensures greater temperature homogeneity within the sample because the cylindrical capillary is completely surrounded by an aluminum cylinder.”
Similarly, U.S. Pat. No. 7,582,429 from the University of Utah Research Foundation provides an overview in paragraph 3 of a number of commercial instruments with melt capabilities: “Various types of thermocyclers have been described in the literature to perform PCR. Some types of thermocyclers with HRM that may be employed with the present embodiments include and are not limited to the AB7300, the HR-1™, the LightCycler 480®, the Master Cycler®, the LightScanner® and the RotorGene™. Each of these instruments typically provides a real time PCR reaction followed by HRM.” However, each of these devices use a heater block in which tubes or capillaries are inserted or feature capillaries that are spun in air as in the Rotor-Gene Q.
Further, U.S. patent application Ser. No. 12/514,671 from the University of Utah Research Foundation describes the typical alternate configuration of melting analysis based on a spatial temperature gradient (i.e., temperature is made intentionally non-uniform).
A high throughput device is desired that creates melt curves that are sufficiently reproducible such that small changes in melt temperature or curve shape can be accurately distinguished. Specifically, the heating system to create these melt curves must have high reproducibility so that small changes in the melt curves can be attributed to deviations in the patient samples (i.e., mutations) rather than merely unwanted deviations in the heating system.
The art describes methods for parallel processing of patient samples using large fixed heater blocks. Throughput is limited by the size of the heater block which holds a fixed number of patient samples and is slow to heat. Reproducibility also suffers when heating blocks are large due to non-uniformity of temperature. Other approaches including those based on capillaries have similar shortcomings in the balance between throughput and reproducibility.
Accordingly, there is a need in the art for a high throughput system that subjects each sample to a controlled and uniform temperature profile.